Mike Clark from the University of Melbourne in Australia presented at London Calling 2019 on “Deep transcriptomic sampling with long-read single cell RNA sequencing.” Clark gave the first talk in the session and explained the power of expression profiles of single cells (scRNA-seq) to identify cell types and variations in gene expression. scRNA-seq can be performed with Illumina sequencing. The 10x scRNA-seq protocol uses barcoded gel beads. The gel beads are processed to cDNA and fragmented for Illumina sequencing. Clark and team have modified the 10x protocol. cDNA from each cell is isolated in “GEMs” and used to construct libraries. Clark split libraries to sequence on both Nanopore and Illumina devices. They used the scPipe workflow: Albacore/Guppy base calling, Fastq trimming, and minimap2 alignment to determine counts. They compared Illumina, MinION, PromethION, and PacBio (2 SMRT flow cells). A comparison of PromethION to Illumina suggests they are obtaining similar amount of reads. Gene counts suggested good correlation between Nanopore and Illumina. Different cell types were separated with all sequencing methods. Clark explained that the next analysis involved the identification of differential isoform expression between cell lines. Clark noted that better tools are needed. I appreciate all the work that went into the multiple comparisons Clark presented!
